The long-range objective of this laboratory is the determination of the detailed mechanisms of enzymatic catalysis, the structure of the protein molecules themselves, and the relationships between structure and function. In the past few years, we have characterized a large number of complexes formed between L-glutamate dehydrogenase and a variety of ligands involved in the reactions it catalyzes. We have studied the interactions between these ligands and have deduced a pattern of eight subsites on the enzyme surface, various combinations of which produce the diverse ligand binding sites. We also know the major features of the transient-state kinetics of the enzyme-catalyzed reaction and the spectroscopic characteristics of the system in the various phases of its time course. We are now ready to turn our attention to the determination of the nature of the events involved in the interconversion of the complexes that constitute the reaction path. We will continue the resolution of the subsites, attempting to learn more of their geometrical relationships and actual chemical identites. We also plan to study the behavior of the system under physiological concentrations of its components and to explore some new techniques for investigating enzyme ligand interactions. We also propose a detailed study by direct microcalorimetry of the specific thermodynamic contributions to ligand binding by the individual enzymatic subsites, with particular emphasis on the thermodynamics of cooperativity.